EFFECT OF MGN-3
ON HUMAN NATURAL KILLER CELL ACTIVITY AND INTERFERON- y SYNTHESIS IN VITRO.
1 - MGN-3-
MGN-3 is an arabinoxylane from rice bran that has been
enzymatically modified by Hyphomycetes mycelia. It is polysaccharides that
contain ß -1.4 xylopyronase hemicellulose. MGN-3 was offered by Daiwa
Pharmaceutical Co.. Ltd. Tokyo. Japan.
2 - Separation of peripheral blood Lymphocytes (PBL):
Mononuclear cells from the subjects were separated from
the fresh whole blood by ficollhypague density gradient centrifugation
(Litton Bionetics, Rockville. MD). The lymphocyte band at the interface
was collected and cells pelleted by centrifugation, washed twice in RPM11640
and suspended in complete medium (CM) which consists of RPMI-1640, supplemented
with 10% (v/v) fetal calf serum. 2mM glutamine, 25 mM Hepes (pH 7.2). 50
units penicillin and 50 ug streptomycin per ml (Grand Island Biologicals.
Santa Clara. CA).
3 - In Vitro Treatment of PBL with MGN-3:
PBL were cultured with MGN-3 at different concentration
(0.25, 50 and 100 ug/ml for 16 hr. PBL was washed twice with CM and adjusted
at I OA- 106 cells I ml.
4-NK Cell Activity:
The most common method of assessing NK anti-Cancer activity
is to use Cr-release assay in which fixed number of Chromium, (CR)- labeled
tumor target cells are incubated with varying number of NK effector cells.
The percent cell mediated lympholysis is calculated from the foillowing
% lysis = Exp. Rel. - Sp- Rel X 100 Total Rel. - Sp. Rel
Where Exp. Rel test refers to the counts in supernatants
from wells containing effector cells. Sp. Rel is the spontaneous release
in medium only, and Total Rel. is the maximum counts obtained by lysis
with detergent. Usually four serial dilution are made of the NK effector
cells and plated in quadruplicate with the tumor targets in microtiter
plates. In brief. 5 x 101 per ml CM. and 0.1 pipetted to quadruplicate
wells to give and effector: Target cell ratio of 100: 1, 50:1, 25:1 and
1 2:1 . Microtiter plates were then centrifuged for 3 minutes at 500 RPM
and following a 4 hours incubation at 37C. and the supernatant were counted
in a gamma counter. The percentage of isotope released was calculated by
the formula mentioned above.
5 - MTT Asaay
Effect of MGN-3 on P388 tumor cell proliferation was examined
by MTT assay. 0.1 n-d -of tumor cells 2xlOl cultured with MGN-3 different
doses in each well of 96 well microtiter plate rounded bottom for one day.
10 li I of MTT (3-(4.5-dimethylthazol-2-yl)- 2.5 diphenyl tetrazolium bromide)
were added to each well for four hours and the plate centrifuged for 10
min. at 2000 RPM. Supernatant was discarded. and 100 ml of acid alcohol
was added to each well. The microtiter plate was read by MR 700 machine.
6 - Assay of I FN- -Y Ploduction:
Peripheral blood lymphocytes (I X
106 CelIS/Ml) were incubated with various concentrations
of MGN-3 (0-200 Mg/ml) for 48 Hr. at 37C under 50% CO, and supernatants
were collected for assay of IFN- y production. The amount of IFN- y in
the supernatants was determined by ELISA using a commercially available
IFN- y ELISA kit from BioSource International (Camarillo. CA).
Results of this study demonstrated that.
1. Treatment with MGN-3 resulted in significant increase
in NK activity post treatment (Figure 1)
2. The mechanism by which MGN-3 augments NK activity through
induction of IFN- y (Figure 2).
3. MGN-3 also possesses direct anti-cancer activity in
vitro. (Figure 3). We Conclude that:
1. MGN-3 is a potent Biological Response Modifier (BRM)
as manifested by highly increase in human NK cell activity at 16 hr. post
2. Earlier work in vivo demonstrated significant increase
in human NK cell activity by MGN-3 at 2 wks post treatment.
3. The mechanism by which MGN-3 induce NK activity is
through production of IFN-'Y
4. MGN-3 possesses direct anti-cancer activity in vitro.
5. The induction of NK activity by MGN-3 may represent
a new immune-therapeutic approach for the treatment of cancer. which needs
to be examined in multiple clinical trials.
EFFECT OF MON-3 ON HUMAN NATURAL KILLER CELL ACTIVITY
AND INTERFERON-y SYNTHESIS IN VITRO.
M.Ghoneum. G. Namatalla and C.Kim. Drew University of
Medicine and Science, Los Angeles, CA 90059, and University of California,
MGN-3 is an extract of arabinoxylane from rice bran that
has been enzymatically treated with an extract from hyphomycetes mycelia.
this study we investigated the effect of MGN-3 on natural killer (NK) cell
activity and interferon- -y (IFN-,y ) synthesis by peripheral blood mononuclear
cells (MNC). MNC was prepared from peripheral blood of healthy individuals
and incubated with various concentrations of MGN-3 for 16 hrs, and then
NK cell activity was measured by 4 hr "Cr-release assay using K562 Tumor
cells as target NK activity was significantly enhanced (2-5 fold) by treatment
with MGN-3 at 0-100 p glml. In an attempt to investigate the mechanism
by which MGN-3 enhances NK activity, we examined the effect of MGN-3 on
IFN- y production by MNC. Culture supernatants of the cells incubated with
MGN-3 were collected and analyzed for INF- -y synthesis by ELISA. UIF-
y production was increased>2 fold. We concluded that MGN- 3 is a potent
biological response modifier (BRM) and may be useful in immunotherapy of
MGN-3. was offered by Daiwa Pharmaceutical Co., Ltd. Tokyo-Japan.